Flavoprotein external monooxygenases (hydroxylases) are important in the catabolism of various organic compounds including drugs, toxicants, and pollutants. Such enzymes activate the molecular oxygen, leading to the insertion of one oxygen atom into a substrate and the reduction of the other oxygen atom to water. With a few exceptions, structural and mechanistic properties of flavo-hydroxylases are generally not well understood. Our long-term goals are to pursue a better understanding of the nature of flavo-hydroxylase catalysis and to delineate the mechanism(s) by which the hydroxylase in vivo activities are regulated. Same as the ongoing project, the present application focuses on the elucidation of flavo-hydroxylase catalysis. Specifically, this proposal is aimed to delineate the structure and mechanism of three selected flavo-hydroxylases, namely bacterial luciferase, salicylate hydroxylase, and the recently isolated 3-hydroxybenzoate-6-hydroxylase. Investigations will be along three lines: (1) As a continuing effort, the kinetic mechanisms of these three hydroxylases will be elucidated further by methods of steady-state kinetics, rapid-mixing techniques, and deuterium and tritium isotope effects. Techniques of rapid scanning and cryoenzymology will also be employed to detect and characterize reaction intermediates. Certain flavin and substrate derivatives and, in the case of luciferase, mutant enzymes which have altered kinetic characteristics will be utilized in the hope that new intermediates may become more readily detectable. A chemical mechanism is proposed as a working model for salicylate hydroxylase. Potential suicide inhibitors will be synthesized and tested as mechanistic probes. (2) Three sulfhydryl-directed/photoaffinity-labeling bifunctional reagents have been synthesized and will be used to "scan" the aldehyde site of luciferase. Additional photoaffinity labelling probes will be developed to determine the subunit location of the luciferase flavin site. (3) The genes for salicylate hydroxylase and 3-hydroxybenzoate-6-hydroxylase will be cloned and sequenced. By translation, the primary structure of the two hydroxylases can then be determined. Through structural and mechanistic studies as those described in this proposal, we hope to obtain a better knowledge of the nature of flavo-hydroxylase catalysis. The gene cloning work will also prepare us for future studies regarding site-specific mutagenesis and regulation of hydroxylase in vivo activities.